Proteolysis by the ubiquitin (Ub) system plays essential roles in a multitude of biological processes, and has major ramifications for human health. Studies of the Ub-dependent N-end rule pathway by this laboratory over the last three decades were made possible, to a large extent, by the present grant (GM031530), currently in its 28th year of supporting our research. A focus of this renewal application stems from the 2010 discovery by the lab that the N-terminal acetylation (Nt-acetylation) of cellular proteins creates specific N-terminal degradation signals (degrons), termed AcN-degrons. The resulting expansion of the N-end rule pathway necessitated the change of the earlier title of this grant ("The Functions, Mechanisms, and Regulation of N-Terminal Arginylation") to its present title, "N-Terminal Acetylation and Protein Degradation by the N-End Rule Pathway". Specific Aims (described more briefly than in Research Plan): 1) Studies of the Nt-acetylation branch of the N-end rule pathway in the yeast Saccharomyces cerevisiae that include the use of recent mass spectrometry-based technologies to identify specific physiological substrates containing AcN-degrons. 2) Further development of the Ub-Reference Technique (URT), a method introduced by this laboratory in 1996. A novel (unpublished) version of URT t allows a nascent protein to undergo modifications (including Nt-acetylation) without kinetic delays. In another project of this Aim, we employ a different technique, based on activation of dormant N-degrons, to tackle a physiologically important problem of the posttranslational (as distinguished from cotranslational) Nt-acetylation. 3) Studies of the Nt-acetylation branch of the N-end rule pathway in mammalian cells. Proteins containing AcN-degrons have not been identified in mammals, until now. We shall focus, initially, on two proteins: Rgs2, a regulator of specific G proteins;and Aanat, the serotonin N-acetyltransferase. Aanat produces Ac-serotonin, the immediate precursor of melatonin, a hormone that regulates circadian rhythm. Regulated degradation of Aanat is likely to be mediated by its AcN-degron. 4) Studies of the previously known part of the N-end rule pathway, now called the UBR/arginylation branch. One of these projects is a study of Atl1 (Ate1's ligand-1) and its complex with Arg-transferase, Ate1. Other studies shall explore the recent (also unpublished) discovery that the UBR branch of the N-end rule pathway is mediated by a physical complex between Ubr1 (the previously known E3 of this pathway) and Ufd4, the E3 of the UFD (Ub-Fusion-Degradation) pathway. Until recently, the UFD pathway, identified and studied by this laboratory in 1986-1995, was thought to be entirely distinct from the N-end rule pathway. PUBLIC HEALTH RELEVANCE: Studies proposed in this renewal application are based on recent discoveries by this laboratory. These studies will address the functions of systems that mediate regulated protein degradation in yeast and mammalian cells. Inborn or acquired defects in these systems, which include the ubiquitin system, are a cause of many human diseases, including cancer and neurodegenerative syndromes. Understanding the mechanisms and functions of the N-end rule pathway, our focus and a major part of the ubiquitin system, is likely to result in better therapies for specific medical problems, including currently intractable ones.